APPLICATION NOTE #20143
Budding Yeast Homogenization:
Reproducible Protein Extracts with Protein Functioned Retained
Eva Paige Syzmanski1, Drexel Neumann2, and Oliver Kerscher1
Integrated Science Center, Biology Department, The College of William and Mary; 2Omni International Inc., Kennesaw, Ga.
1
Figure 2
This Application Note was developed based
with the approval of Eva Paige Syzmanski
and Oliver Kerscher, based off their
publication in the Journal of Visualized
Experiments (2013) doi: 10.3791/50921.
Saccharomyces cerevisiae has historically
been heralded as an excellent model
organism in molecular research due to its
quick reproduction cycle, large population
study abilities, ease of growth, and
well documented genome1, 2. Therefore
this common baker’s yeast is a familiar
organism in many research labs.
Despite the great research benefits of the
organism, many complications arise when
working with the yeast due to a robust
cell wall3. The yeast cell wall is a highly
evolved organelle that is adaptive and
responsive to environmental stress. The
effectiveness of the strong, but elastic
cell wall is highlighted by its preservation
across many other yeast species. Although
beneficial for the organism, the cell wall
proves to be an obstacle for researchers
interested in analyzing its molecular
components due to its difficult disruption.
Bead mill homogenization, as performed
by the Bead Ruptor Elite, offers a robust
and quick method for disrupting yeast
cells. Bead mills are particularly attractive
because of the automated nature of their
operation, eliminating variation caused
by human error. Their ability to process
a large number of samples at once
eliminates sample processing bottlenecks,
creating the potential for high throughput
studies. Omni International’s Bead Ruptor
Elite (P/N’s: 19-040E; 19-010-310) provides
researchers with a rapid, efficient, and
reliable method for disrupting yeast
Figure 1 Budding Yeast
cells in a sterile and controlled manner.
Despite the vigorous nature of bead mill
homogenization, downstream molecular
analyses methods are unaffected and
samples are open to a variety of methods
including PAGE, Western blotting,
protein functional studies, and nucleic
acid purification. Herein we describe the
methods used by Szymanski and Kerscher
to disrupt yeast cells for western blotting
and protein functional studies.
Methods
Yeast cultures were transformed with
plasmids carrying a 6xHIS V5-tagged
SUMO ligase Siz1, GST tagged Slx5 and a
HA tagged Siz1∆440. Cells were isolated
by centrifugation and resuspended in
PBS with a protease inhibitor cocktail.
Following resuspension, cells were
prepared for storage or downstream
analysis by high-speed centrifugation and
snap-freezing in liquid nitrogen.
Frozen pellets were added to 2 mL tubes
containing 0.5 mm glass beads (P/N:
Figure 3
Figure 2 SDS-PAGE stained with Coomassie of
whole cell extracts demonstrating reproducible
protein recoveries.
Figure 3 Western Blot of Siz1 shows no protein
degradation or shearing.
19-622) and 500 µL of an ice-cold lysis
buffer. Samples were homogenized
through bead beating in a Bead Ruptor
24 at 5.5 m/s for 20 seconds, six times,
with 1 minute incubation on slushy ice
between each run. Homogenates were
clarified by centrifugation then protein
precipitation was performed by addition
of TCA. Protein pellets were resuspended
and stored at -80 °C for future use. Protein
samples were separated by SDS-PAGE to
demonstrate the reproducibility of the
protein extractions (figure 2). Western
blotting was then performed using an
anti-V5 antibody to demonstrate the
presence of the V5-tagged SUMO ligase
Siz1 in the whole cell lysate (figure 3)
Syzmanski and Kerscher further evaluated
if bead milling would impact a proteins
tertiary structure and thus its function.
continued
Methods (continued)
Figure 4
Figure 4 Western Blot for
SIx5 and Siz1∆440 shows
recovery following resin
based purification under
native conditions, but not
denaturing conditions.
Slx5 and Siz1∆440 both possess a RING
domain that has been reported to
naturally coordinate with Zn2+ ions.
Both proteins were suspended in either
a non-denaturing or denaturing buffer
then subjected to affinity capture
through immobilized metal affinity
chromatography. The captured proteins
were then eluted and separated via
1D-PAGE and detected through western
blotting using either an anti-GST or antiHA antibody.The integrity of the proteins
tertiary structure was demonstrated by
the fact that the protein was detected
under native conditions but absent under
denaturing conditions (figure 4).
Conclusions
Syzmanski and Kerscher’s study
established a foundation of procedures
from which future yeast research
can be built upon. In their study they
show the benefit of using bead mill
homogenization as a method to efficiently
disrupt yeast in a reproducible and
conservative manner. This technology
provides researchers with a fast, user
friendly mechanism to perform highthroughput studies, eliminating sample
prep bottlenecking.
Bead Ruptor – 2 mL Carriage Kit: 19-010-310
References
0.5 mm Glass Bead Kit: 19-622
1.
Botstein D, Chervitz SA, & Cherry JM.
(1997). Yeast as a Model Organism.
Science. 277(5330): 1259 – 1260.
2.
Ashburner et al. (2000). Gene
Ontology: tool for the unification of
biology. Nature Genetics. 25: 25 – 29.
3.
Klis FM, Boorsma A, & De Groot PWJ.
(2006). Cell wall construction in
Saccharomyces cerevisiae. Yeast. 23:
185 – 202.
Part Numbers Referenced
Bead Ruptor Elite Motor Unit: 19-040E
Bead Ruptor Elite
935-C Cobb Place Blvd. NW
Kennesaw, GA 30144
800.776.4431 • 770.421.0058
www.omni-inc.com